Hexasodium 4,4'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis[5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate]

Endpoint summary

Additional information

Ames test:

In a reverse gene mutation assay the bacteria strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium  were exposed to the test substance (80% act. ingredient) at five concentrations in the range of 61.7 to 5000 µg/plate in the presence and absence of a metabolic activation system applying the standard plate incorporation method. The rat-liver post mitochondrial supernatant (S9 fraction) was used as an extrinsic metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentration range. Each strain was additionally tested in the presence and in the absence of the metabolic activation system with a suitable, known mutagen as positive control. The results indicated no increase in the incidence of histidine-prototrophic mutants in comparison with the negative control. In the mutagenicity tests normal backround growth was observed with all strains at all concentrations. The numbers of revertant colonies were reduced at the upper concentrations. The test substance thus exerted a slight inhibitory effect on the growth of the bacteria.

MNT in vitro:

In the key study (BASF, 2015), the test item (purity: 88.0 g/100 g), dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA, the exposure period was 20 hours without S9 mix. In Experiment IIB, the exposure period was 4 hours with S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 2273.0 μg/mL was chosen with regard to the correction factor of 1.14 of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH was observed. In Experiment I in the absence and presence of S9 mix and in Experiment IIB in the presence of S9 mix, no cytotoxicity was observed up to the highest required concentration. In Experiment IIA in the absence of S9 mix after continuous treatment the highest applied concentration was not evaluable for cytogenetic damage due to strong cytotoxic effects. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment IIB in the presence of S9 mix one statistically significant increase in the number of micronucleated cells (1.25 %) was observed at a concentration of 742.2 μg/mL. The value was within the laboratory historical solvent control data (0.15 – 1.70 % micronucleated cells) and additionally no dose dependency could be observed. Therefore the finding can be considered as biologically irrelevant. Either Demecolcin (50.0 ng/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentration.

In a second study (BASF, 2015), the reproducibility of the evaluable concentrations due to toxic effects and the outcome of the study were very inconsistent (nearly all evaluated maximal concentration showed toxic effects taking the relative value of the cell counts compared to the solvent (< 0.5); these observed toxic effects were not reflected by the RICC or PI; the RICC value showed a huge fluctuation between the experiments and single concentration, based on the experimental protocol used (not very robust); the values calculated for RICC did not give clear information about the toxicity of the test item, therefore the absolute cell numbers were reported additionally; these relative values showed a clear and dose dependent toxicity in all experiments). Therefore, this study was disregarded.

HPRT test:

The study was performed to investigate the potential of the test substance (purity: 88.0 g/100 g) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. Based on a calculation error 1900 μg/mL instead of 5680 μg/mL (equal to 5000 μg/mL of the pure substance, regarding the content (88.0 g/100 g, corresponding to a correction factor of approx. 1.14) was chosen as maximum concentration of the range finding pre-experiment. The concentration of the second experiment was based on cytotoxicity data generated in the first experiment. The test item was dissolved in deionised water. Test item concentrations between 14.8 μg/mL and 1900 μg/mL were used. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I at 237.5 μg/mL and above without metabolic activation and at 356.3 μg/mL and above with metabolic activation. In experiment II cytotoxic effects as described above were noted at 180.0 μg/mL and above without metabolic activation and at 420.0 μg/mL with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells und thus is considered to be non-mutagenic in this HPRT assay.

Justification for selection of genetic toxicity endpoint
GLP and guideline conform studies. The studies fulfill the REACH information requirements of Annex VIII, section 8.4.

Short description of key information:
The test item indicated no increase in the mutation frequency in bacteria (Ames test), did not induce gene mutations at the HPRT locus in V79 cells (HPRT assay) and did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be genotoxic under Regulation (EC) No 1272/2008,as amended for the seventh time in Regulation (EC) No 2015/1221.